ABSTRACT
Studies were made of the effects of Picloram at various
concentrations on whole water samples and cultures established
from tows from Monterey Bay, California, using the C and
oxygen light/dark bottle techniques. The rate of net
photosynthesis was depressed approximately 10% by a herbicide
concentration of one part per million by weight and 89% by a
herbicide concentration of one hundred parts per million by
weight. No significant effects of the herbicide on respiration
was detected at these concentrations.
-1-
INTRODUCTION
The purpose of this study was to investigate the effects
of the herbicide Picloram on rates of photosynthesis and
respiration in the natural marine phytoplankton community
found in Monterey Bay, California, during May, 1969. It was
hypothesized that photosynthetic rates would be depressed
at concentrations in parts per billion by weight, and that
respiration rates would be increased at similar concentrations.
Picloram, or Tordon, as it is commercially known, is a
herbicide with a half-life of up to a year. Usually, its
targets are perennial, broad-leaved plants and stands of
brush, in which it accumulates in new growth. Although
used frequently as a soil sterilant, it is also used to
eliminate growing plants. Despite the fact that Picloram
was not used in Monterey County during 1968 (because of its
residual nature and because the entire supply was purchased
by the United States government), Picloram was chosen for
this study because of its use as a defoliant by the United
States in Vietnam, where application rates may be double to
quadruple those in the U.S. At such high application rates,
monsoon rains might raise herbicide concentrations in lakes
and inshore waters of Southeast Asia to levels which might
significantly effect phytoplankton primary production over
long periods of time.
9
-3-
My thanks go to Dr. Malvern Gilmartin, Dr. Isabella A.
Abbott, and Dr. John H. Phillips, Jr., for their invaluable
advice. In addition, I am indebted to Mr. Harry Agammalion
for providing me with a supply of Picloram and much information
regarding its use.
This work was supported in part by the Undergraduate
Research Participation Program of the National Science
Foundation, Grant 464-5878.
MATERIALS AND METHODS
Picloram:
Ihe herbicide was in a commercial formulation called
Tordon 22K and dissolved in acetone at a concentration of
2.36 x 10—+ milligrams/milliliter. It was diluted with
filtered (0.45 micron millipored) sea water. At concentrations
of approximately 2.36 x 10  milligrams/milliliter and
above, a calcium salt precipitate in sea water was noted.
Acetone controls were run parallel to those concentrations
which were found to effect photosynthesis and respiration.
Sampling:
Samples were taken almost daily from the same location
in Monterey Bay. Whole water samples were taken at a depth
of approximately 124 meters with a 5 liter Van Dorn sampling
3/0
device (Van Dorn 1956). The sea water was either directly
siphoned from the Van Dorn into the BOD bottles to be used
for determinations of photosynthetic rate, or, alternately
transferred into a 5 gallon plastic carbuoy which was brought
back to the laboratory, where siphoning into BOD bottles was
immediately begun.
Cultures were established using tows made with a number
25 phytoplankton net at approximately the same depth. Un
returning to the laboratory, the tows were immediately diluted
with enriched sea water (Institute of Marine Resources culture
medium). They were kept in 2 liter fernbach flasks in a water
bathsof 15 degrees centigrade with constant lighting.
Samples were preserved from tows,and dominant phytoplankton
identified every three days. The phytoplankton community
composition is presented in Table I.
Measurements:
Measurements of photosynthesis were made using the
oxygen light/dark bottle (Gran 1927) and C1" (Doty 1954)
techniques. Dissolved oxygen was determined by a modified
Winkler method (Parsons and Strickland 1968). Clear 300
milliliter biological oxygen demand bottles were used for
culture flasks. Acetone controls at the concentrations which
affected photosynthesis were run in the same size bottles and
measured by radioactive carbon uptake. They were inoculated
with 10 milliliters of natural composition culture and filled
with filtered (0.45 micron millipored) sea water. C uptake
was similar in whole water controls to that in inoculated
controls, so that cell concentrations were assumed to be
similar. All BOD bottles were directly inoculated with the
appropriate amount and concentration of Picloram or acetone.
Clear bottles were incubated for 6 hours in a Doty
incubator (Doty 1959) and dark bottles in a nearby chamber.
Ihe temperature of water surrounding the bottles was maintained
at approximately 15 degrees centigrade.
Measurements of respiration were made by following the
decrease in dissolved oxygen in darkened 60 milliliter
glass-stoppered bottles. These were inoculated with 20
milliliters of natural composition culture, siphoned full
of filtered (0.45 micron millipored) sea water, and injected
with the appropriate amount of herbicide.or acetone. hey
were incubated for 6 hours at a temperature of about 15 degrees
centigrade.
RESULTS
Measurements of gross primary production were first made
by determining the amounts of dissolved oxygen in light and
dark bottles after incubation in four concentrations of
Picloram, O.OOl parts per billion, O.1 ppb, 10 ppb, and 1000 ppb.
When variable results were obtained, a fifth concentration of
100,000 ppb was added. These results are presented in Table
II.
Oxygen and ++ uptake determinations were used to measure
photosynthetic rate at concentrations which appeared to effect
the phytoplankters. Since C uptake measures a value much
closer to net than gross primary production, net primary
production was determined using dissolved oxygen measurements.
These results are presented in Tables III and IV.
Acetone controls were run at concentrations parallel to
those which affected photosynthesis using C uptake measurements
These results are presented in Table V.
Respiration was measured at selected concentrations
affecting photosynthesis, by determining decrease in dissolved
oxygen. Acetone controls were run parallel to the Ficloram
determinations. These results are presented in Tables VI and
VII.
DISCUSSION
Ihe results from the initial oxygen determinations of
gross primary production (see figure 1), although far too
variable to be statistically significant, the results suggested
a depression of photosynthesis at 1000 ppb and 100,000 ppb.
Determinations of net primary production at the higher
5
concentrations of Picloram indicate beyond doubt that photosyn-
thesis is depressed at a concentration of 1000 ppb and almost
eliminated at a concentration of 100,000 ppb (see figure 2).
However, photosynthesis is also somewhat depressed by acetone
at those concentrations. The relative depression of photo¬
synthesis by Picloram and acetone indicate that Picloram has
a distinct effect beyond the acetone in which it is dissolved.
However, one must consider the possibility that phytoplankton
incubated in acetone may not react the same way to Picloram
as healthy phytoplankton.
Respiration determinations (see figure 3) again show too
much variability to be statistically significant. Although
uncorrected for bacterial respiration, the means indicate that
respiration rate is increased, but probably because of incubation
in acetone.
Such results indicate that Picloram acts primarily on the
chlorophyll or some other substance involved only in photosynthesis,
as opposed to other pesticides, like DDT, which affect both
respiration and photosynthesis, and might therefore be affecting
membrane structure.
Table I. Phytoplankton Community Composition
Predominant phytoplankters
Date
5/13
Coscinodiscus
(diatom)
5/16
Coscinodiscus (diatom)
Schroederella (diatom)
Lyngbya (blue-green alga)
Coscinodiscus (diatom)
5/19
Lyngbya and other blue-green algae
Rhizosolenia alata (diatom)
5/22
Rhizosolenia hebetata (diatom)
Chaetoceros spp. (diatom)
Nitzschia pacifica (diatom)
Rhizosolenia alata (diatom)
5/26
Rhizosolenia hebetata (diatom,
Chaetoceros spp. (diatom)
Nitzschia pacifica (diatom)
Skeletonema (diatom)
3
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C
-10
Percent Net Primary Production after incubation
Table III.
in Picloram.
Oxygen light/dark bottle method.
Picloram Concentrations
1000 ppb 10,000 ppb 100,000 ppb
Control
Expt + Date
5/21
122.92
37.50
32.29
100.00
2a
-35.95
100.00
59.84
16.00
-1.83
100.00
91.38
26.75
Means:
Maximum Control Variability: 32.29%.
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285
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Table VI. Percent Respiration after incubation in Picloram.
Oxygen light/dark bottle method.
Picloram Concentrations
1000 ppb 10,000 ppb 100,000 ppb
Control
Expt + Date
83.33
156.25
170.83
5/27
100.00
Sa
168.47
124.45
100.00
73.92
5/28
5b
24.07*
267.05*
100.00
384.74*
5/30
5c
78.66
139.75
100.00
103.35
5d
5/31
*Not included on graphs because of poor experimental control.
-14-
Table VII. Percent Respiration after incubation in Acetone.
Oxygen light/dark bottle method.
Picloram Concentrations
which correspond to
Acetone Controls.
1000 ppb 10,000 ppb 100,000 ppb
Expt + Date
Control
106.25
131.25
191.67
100.00
5a
5/27
121.19
117.97
100.00
78.81
5b
5/28
380.94*
365.76*
100.00*
-40.85
5c
5/30
178.66
56.90
100.00
43.93
5/31
5d
*Not included on graphs because of poor experimental control
100.
80
2


60
E



40
—



20



0.1
0 1000 100,000
0.001
PICLORAM CONCENTRATION IN PARTS PER
BILLION
Percent Gross Primary Production after
Fig. 1.
incubation in Picloram. Sea water controls
are set at 100.00%. Each point is the mean
of 6 bottles.
26
16

1001
80
T 44
60

EXPT 48
CORRESPONDING ACETONE CONCENTRATIONS
004
—
3
80



60





EXPT 24
——EXPI
34
E
2 20
—-EXPT 36

5000 10,000 50,000 100,000
1000
PICLORAM CONCENTRATION IN PARTS PER
BILLION
Percent Net Primary Production after incubation
Fig. 2.
in Picloram or Acetone. Sea water controls are
set to 100.00%. Each point is the mean of 2
bottles.
-17-
190
160
130

100
70
40
CORRESPONDING ACETONE CONCENTRATIONS
190
160
2

130


œ 100
S
70
)
5
2—

1000
10,000
100,000
PICLORAM CONCENTRATION IN PARTS PER
BILLION
Percent Respiration after incubation in
Fig. 3.
Picloram or acetone. Sea water controls are
set at 100.00%. Each point represents 1
bottle.
REFERENCES
Doty,
M.S. 1954. Current Status of Carbon Fourteen Method
of Assaying Productivity of the Ocean. Univ. Hawaii
Annual Rep.: 1-42.
M.S. and M. Oguri. 1959. Carbon Fourteen Technique for
Doty,
determining Primary Plankton Productivity. Pubbl. Staz.
Zool. Napoli. 31 Suppl.: 70-94.
Gran, H.H. 1927. The Production of Plankton in the Coastal
Waters off Bergen, March-April 1922. Fiskeridir. Skr.
Fisk. 3(8): 1-74.
Strickland J.D.H. and T.R. Parsons. 1968. A Practical Handbook
of Seawater Analysis. Bull. Fish. Res. Bd. Can. 167: 21-
26, 261-278.
Van Dorn, W.G. 1956. Large Volume Water Samplers. Tran. Am.
Geo: Union. 37: 682-684.
1967. Herbicide Handbook of the Weed Society of
America.: 12-16.
200
50
100
2
50
— 200

C
150
O
O.
L
D00
50
Fig. 3.
17.

ACE


--- EXPT 50
2— EXPT 5b
— EXPT 54
1000
10,000
100000
DCLORAN
P
Percent Respiration after incubation in
Picoram or Acetone. Sea water controls are
set at 100.00%. Each point represents 1
bottle.
2
C
—
S
C
—
O

L
5
Fig. 2.
00
80
60
1OO
80
60
40
20




—A EXPT 40
EXPT 4b
ACE
S
------ EXPT 20
—A EXPT30
—EXPT 3b

10,000 50,000 100,000
1000 5000
H.D
ICLOR/
Percent Net Primary Production after incubation
in Picloram or Acetone. Sea water controls are
set at 100.00%. Each point is the mean of 2
bottles.
Z
C
2
O
E
O
0)
0
C
O
o
Fig. 1.
100
80
60
40
20
-15-
1000 100,000

(
ICLORAT
Percent Gross Primary Production after
incubation in Picloram. Sea water controls
are set at 100.00%. Each point is the mean
of 6 bottles.
33